This invention relates to the field of labels, particularly labels for diagnostic or analytical use. In particular, it relates to oligonucleotides that are modified to enhance the binding affinity or the binding specificity of the oligonucleotides for complementary sequences and that in addition optionally bear a readily detectable characteristic.
Sequence specific binding of oligonucleotides both to single stranded RNA and DNA and to duplex DNA is widely known. This phenomenon has been harnessed for a great variety of diagnostic, therapeutic and analytical, e.g., sequence determination or gene mapping, purposes. Previously, one objective of research in this field has been to increase the affinity of such oligonucleotides for their complementary sequences. For example, workers have described oligonucleotides containing 5-substituted pyrimidine bases that substantially increase the Tm for oligonucleotide binding to complementary bases (International Publication No. WO 93/10820).
Publications have described the use of fluorescent cytosine derivatives to prepare labeled DNA probes. See Inoue et al., Jpn. Kokai JP 62059293, (1987). In addition, fluorescent labeled nucleotides have been employed in DNA sequencing. See Prober et al., xe2x80x9cSciencexe2x80x9d 2:336-341 (1987).
1,3-Dihydro-2H-imidazo[4,5-b]-quinolin-2-one derivatives as phosphodiesterase inhibitors are disclosed by Raeymaekers et al. (EP 541,153).
U.S. Pat. No. 5,502,177, discloses phenoxazine polycycle-containing oligonucleotides and monomers for preparing the oligonucleotides.
The invention compositions or methods accomplish one or more of the following objects.
An object of this invention is to increase the affinity of oligonucleotides for their complementary sequences.
An object of this invention is to increase the specificity of oligonucleotides for their complementary sequences.
Another object of this invention is to provide detectable labels for use in diagnostic assays.
Another object is to enhance diagnostic assays that use oligonucleotides.
Another object is to improve the therapeutic efficacy of oligonucleotides.
Another object is to improve the potency of oligonucleotides as antisense reagents that affect gene expression by altering intracellular metabolism of complementary RNA sequences encoding a target gene(s).
Another object is to provide chemical intermediates and synthesis methods to prepare the invention compositions.
These and other objects of the invention will be apparent when one considers the disclosure as a whole.
In accordance with the objects, the invention provides compounds having the structure (1) 
and tautomers, solvates and salts thereof, wherein
R1 is a binding partner, a protecting group, a linker or xe2x80x94H;
R2 is A(Z)X1, wherein A is a spacer and Z independently is a label bonding group optionally bonded to a detectable label, but R2 is not amine, protected amine, nitro or cyano;
R27 is independently xe2x80x94CHxe2x95x90, xe2x80x94Nxe2x95x90, xe2x80x94C(C1-8 alkyl)xe2x95x90 or xe2x80x94C(halogen)=, but no adjacent R27 are both xe2x80x94Nxe2x95x90, or two adjacent R27 are taken together to form a ring having the structure, 
where Ra is independently xe2x80x94CHxe2x95x90, xe2x80x94Nxe2x95x90, xe2x80x94C(C1-8 alkyl)= or xe2x80x94C(halogen)=, but no adjacent Ra are both xe2x80x94Nxe2x95x90;
R34 is xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94 or xe2x80x94N(CH3)xe2x80x94; and
X1 is 1, 2 or 3.
When the binding partner R1 is an oligonucleotide, embodiments of the compounds of this invention include oligonucleotides of structure (2), (2A), (2B), or (2C) 
wherein
D is xe2x80x94OH, protected xe2x80x94OH, an oligonucleotide coupling group or a solid support;
D1 is an oligonudeotide coupling group, xe2x80x94OH, protected xe2x80x94OH or a solid support, wherein D1 is bonded to one 2xe2x80x2 or 3xe2x80x2 position in the oligonucleotide of structure (2) and the adjacent 2xe2x80x2 or 3xe2x80x2 position in structure (2) is substituted with R21, provided that D and D1 are not both an oligonucleotide coupling group or they are not both a solid support;
D2 is xe2x80x94CO2R5, xe2x80x94C(O)N(R5)2, xe2x80x94SO3R5, xe2x80x94SO2N(R5)2 or an activated derivative of xe2x80x94CO2H or xe2x80x94SO3H;
D3 is a protecting group, xe2x80x94H or xe2x80x94(CH2)2-6xe2x80x94N(R5)2;
R4 is independently a phosphodiester linkage or a phosphodiester substitute linkage, wherein R4 is bonded to one 2xe2x80x2 or 3xe2x80x2 position in the structure (2) oligonucleotide and the adjacent 2xe2x80x2 or 3xe2x80x2 position in structure (2) is substituted with R21;
R5 is independently xe2x80x94H or a protecting group;
R21 is independently xe2x80x94H, xe2x80x94OH, halogen or a moiety that enhances the oligonucleotide against nuclease cleavage;
R37 is independently xe2x80x94Oxe2x80x94, xe2x80x94CH2xe2x80x94 or xe2x80x94CF2xe2x80x94;
n is an integer from 0 to 98; and
B independently is a purine or pyrimidine base or a protected derivative thereof, provided that at least one B is a base of structure (3) 
Embodiments indude compositions useful as intermediates in making the structure (1) compounds, including intermediates having structure (4) 
and tautomers, solvates and salts thereof wherein,
R24 is a halogen; and
R25 is xe2x80x94SH, xe2x80x94OH, xe2x95x90S or xe2x95x90O.
In a further embodiment, the invention includes contacting a structure (2), (2A), (2B), or (2C) oligonudeotide, wherein n is at least about 7, with a sample suspected to contain a nucleic acid having a base sequence that is at least substantially complementary to the structure (2), (2A), (2B), or (2C) oligonucleotide.
In a further embodiment, the invention includes detecting the presence, absence or amount of a complex comprising a structure (2), (2A), (2B), or (2C) oligonucleotide, wherein n is at least about 7, and a nucleic acid having a base sequence that is at least substantially complementary to the structure (2), (2A), (2B), or (2C) oligonucleotide.
In a further embodiment, the invention includes converting a structure (4) compound to a compound of structure (1) where R34 is xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94 and the R2 atom or moiety alpha to the ring containing R27 is xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94 or xe2x80x94CH2xe2x80x94, by displacing R24.
In a further embodiment, the invention includes converting a structure (4A) compound (a structure (1) compound where R2 is replaced with xe2x80x94NH2); to a compound of structure (1), by reductive alkylation of the xe2x80x94NH2 group to yield a structure (1) compound where the R2 moiety alpha to the ring containing R27 is xe2x80x94NHxe2x80x94.